Project Summary/Abstract: An important factor in the control of gene regulation is the 3-dimensional organization of the nucleus, which is dynamically assembled and regulated in different cellular states. Yet, how this nuclear organization is established and how it dynamically changes across cell states is largely unknown. Recently, several nuclear- retained long non-coding RNAs (lncRNAs) have been shown to be important for shaping 3-dimensional genome organization. It is currently unknown how many of the thousands of chromatin-associated lncRNAs may similarly be important for shaping nuclear organization. The main challenge in addressing this question is that we currently lack the tools to comprehensively integrate RNA into our understanding of genome organization. Current genome-wide methods for mapping RNA-DNA interactions can only map a single RNA at a time, do not provide information about the 3-dimensional interaction of their targets, provide an ensemble view derived from millions of cells, and provide an aggregate picture across all RNA molecules, rather than single molecule resolution of RNA localization in the nucleus. Here, we aim to develop a novel approach to enable comprehensive single molecule mapping of the 3-dimensional DNA targets of all RNA molecules within thousands of single nuclei. First, we will a develop a novel genome-wide sequencing method that will enable the generation of comprehensive single molecule maps of the 3-dimensional DNA targets of all RNA molecules in the nucleus (Aim 1). Next, we will develop novel microfluidic devices that will enable the measurement of RNA-DNA nuclear compartments in hundreds to thousands of individual nuclei (Aim 2). Finally, we will extend this technology to study the single cell temporal dynamics of RNA-DNA interactions across cellular reprogramming and functionally validate these maps by testing the role of several identified RNAs in nuclear organization (Aim 3). These methods will overcome a major barrier by enabling, for the first time, comprehensive exploration of the role of nuclear-retained RNAs in shaping nuclear structure. Furthermore, these methods will provide transformative tools for studying nuclear structure ? beyond the immediate questions explored in this proposal, we expect that these tools will be generally applicable to many additional questions for understanding nuclear structure in single cells.